cd47 antibody-induced engulfment of human leukaemic t-cells … · 2017-07-10 · g. lovell, c....
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CD47 antibody-induced engulfment of human leukaemic T-cells by bone-marrow derived macrophagesG. Lovell, C. Szybut, K. Patel, H. Campwala, N. Bevan, D. Appledorn, T. Dale & D. Trezise
Essen BioScience, Welwyn Garden City, AL7 3AX UK or Ann Arbor, MI, 48108, USA
www.essenbioscience.com
Anti-CD47 engulfment does not involve target cell apoptosis
T-lymphoblast CCRF-CEM (1.5 x104
cells/well) were incubated with 5µg/ml anti-CD47 for 1 hour. Thesetreated cells were then added into a96-well micro-titre plate containingbone marrow derived macrophages(BMDM) seeded at 1 x104 cells/well.The plate was placed in an IncuCyte®live-cell analysis system within astandard cell incubator and phaseand fluorescence images werecaptured every 15-30 minutes at 20xmagnification.
Labelled CCRF-CEM cells were treated with increasing concentrations of anti-CD47 antibody (B6H12.2, Abcam; ab3283)and added to human bone marrow-derived macrophages (BMDM; values shown are mean ± SEM, n=4).
• Target cells are labelled using the IncuCyte pHrodo Red Cell Labelling Kit which covalently attaches a pH-sensitivedye to proteins on the exterior of the cell.
• Labelled target cells are added to a standard 96-well microtitre plate containing effector phagocytes (e.g.macrophages, dendritic cells), then placed in an IncuCyte live cell analysis system within an incubator.
• Phase contrast and fluorescence images are taken at regular intervals.
• Fluorescence (area or intensity) increases as the target cells are engulfed into the effector cell lysosome (low pH).
• A kinetic profile showing real-time cell engulfment is generated using the IncuCyte software.
• Inhibitors with varying mechanisms of action (actin polymerisation inhibitor, microtubule disruptor, PKC inhibitor)were applied to the effector cells (J774A.1 mouse macrophages).
• Four concentration-response curves generated (triplicate data, 7 point curves) from a single 96-well plate.
Fluorescence intensity
Object count
• Engulfed cells are observed inside the macrophages and have high fluorescence intensity.• The fluorescence (total fluorescent area or intensity) is proportional to the amount of cell phagocytosis.• Rapid imaging (2 minute intervals) can be performed to create movies showing individual engulfment events.
Phagocytosis ApoptosisAnnexin V
ApoptosisCaspase 3/7
Continuous Live-Cell Analysis: Methodology
• CD47 is a cell-surface marker of self, or “don’t-eat-me” signal. Expression of CD47 enables tumour cellsto evade clearance by host macrophages.
• Blocking CD47 holds great promise as a therapeuticstrategy for both solid and hematologic cancers.
• Here, we have developed and validated anautomated image-based 96-well assay for anti-CD47antibody-mediated cellular phagocytosis.
• Tumour cells are labelled with pHrodo, a pH-sensitivedye, and added to phagocytes (e.g. BMDMs).
• When the labelled cell is engulfed into the acidiclysosome; the change in pH causes the labelled cellsto become fluorescent.
• The approach is amenable to many cell types andenables researchers to screen for small molecules orantibodies that enhance or inhibit phagocytosis.
Overview
Phagocytosis time-course is effector-cell dependent
Automated 96-well assay and analysis
Imaging and quantification
Anti-CD47 Ab induces phagocytosis of CCRF cells
Anti-CD47 directly inhibits proliferation at later time points
Jurkat CCRF-CEM Neutrophils
J774A.1
RAW264.7 NT NT
THP-1 NT
BMDM NT
Effe
cto
r
Target
Confluence at 3h Confluence at 24h
• Anti-CD47 antibody induces phagocytosis of CCRF-CEM cells by J774A.1 mouse macrophages without inducingapoptosis. Data shown are for 5 µg ml-1 anti-CD47- and camptothecin-treated (apoptotic) CCRF-CEM cells.
Cellular phagocytosis assay principle
Validation of labelling step:
• pHrodo-labelled Jurkat cells display red fluorescence in low pH buffer (pH4).
• Fluorescence intensity and the number of cells (objects) exceeding threshold brightness were determined using an IncuCyte live-cell analysis system.
• Intensity increase is proportional to the label concentration.
• The number of labelled cells exceeding the intensity analysis threshold plateaus.
• Anti-CD47 antibody exerts a direct anti-proliferative effect on CCRF-CEM cells in mono-culture; no effect observedafter 3 h, whereas a marked reduction seen at 24 h. Data shown are for 5 µg ml-1 anti-CD47- and camptothecin-treated (apoptotic) CCRF-CEM cells.
Proliferation
Differential time-courses observed for anti-CD47-induced phagocytosis in different effector cells.
• Exemplified target:effector combinations.
63
125
250
500
1000
0 .0
0 .5
1 .0
1 .5
[p H ro d o ] (n g /m L )
Inte
gra
ted
Re
d I
nte
ns
ity
x1
08
(RC
U x
m
2/i
ma
ge
)
63
125
250
500
1000
0 .0
0 .5
1 .0
1 .5
[p H ro d o ] (n g /m L )
Ob
jec
t C
ou
nt
x1
03
(1/m
m2)
Veh
icle
IgG
An
ti-C
D47
Ap
op
tot i
c
0
1
2
3
4
5
Re
d O
bje
ct
Are
a x
10
3(
m2)
Veh
icle
IgG
An
ti-C
D47
Ap
op
tot i
c
0
4
8
1 2
1 6
Gre
en
Ob
jec
t A
re
a
(% c
on
flu
en
ce
)
Veh
icle
IgG
An
ti-C
D47
Ap
op
tot i
c
0
4 0 0
8 0 0
1 2 0 0
1 6 0 0
Gre
en
Ob
jec
t C
ou
nt
(1/m
m2)
Veh
icle
IgG
An
ti-C
D47
Ap
op
tot i
c
2 0
2 5
3 0
3 5
4 0
4 5
Ph
as
e a
re
a(%
co
nfl
ue
nc
e)
Veh
icle
IgG
An
ti-C
D47
Ap
op
tot i
c
2 0
2 5
3 0
3 5
4 0
4 5
Ph
as
e a
re
a(%
co
nfl
ue
nc
e)
0 6 1 2 1 8 2 4
2 0
2 5
3 0
3 5
4 0
4 5
T im e (h)
Ph
as
e a
re
a(%
co
nfl
ue
nc
e) V e h ic le
IgG
A n ti-C D 4 7
C a m p to th e c in
0 4 8 1 2
0
1
2
3
4
5
T im e (h)
Re
d O
bje
ct
Are
a x
10
3
(m
2/i
ma
ge
)
B M D M ; a n ti-C D 4 7
B M D M ; IgG
J 7 7 4 A .1 ; Ig G
J 7 7 4 A .1 ; a n ti-C D 4 7
0 6 1 2 1 8 2 4
0
5
1 0
1 5
2 0
2 5
T im e (h)
Re
d O
bje
ct
Are
a x
10
3(µ
m²/
Ima
ge
)
6 .2 5 : 1 1 2 .5 : 1 2 5 : 1 5 0 : 1
0
2
4
6
8
1 0
J u rk a t : J 7 7 4 A .1 ra tio
AU
C x
10
5 (
0 -
44
h)